Atrial natriuretic peptide (ANP) is a cardiac hormone that regulates sodium excretion, fluid volume, and vasorelaxation, important factors in the control of blood pressure and blood volume. Interaction of ANP with its receptor guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) produces the intracellular second messenger cGMP, which plays a central role in the pathophysiology of hypertension and cardiovascular disorders. Gaining insight into the intricacies of ANP/NPRA/cGMP signaling pathways is of pivotal importance for understanding both receptor biology and the disease state arising from abnormal hormone-receptor interplay. The long-term objective of this proposal is directed at elucidating the nature and mode of functioning of NPRA at the molecular level. The insights gained from domain mapping of cDNA clones and biochemical analysis of the receptor will be used to elucidate those receptor areas that can be further analyzed by site- directed mutagenesis in transfected cells in vitro and transgenic mouse models in vivo to learn what structural components are involved in the functioning of NPRA. This proposal has four major, intimately linked goals, all involving a primary focus on NPRA as an approach to gain increased understanding of the normal and abnormal control of cellular and physiological processes. These goals are as follows: 1) delineate the molecular determinants in the GC catalytic active-site of NPRA by site-directed mutagenesis and determine the role of ANP/NPRA signaling and physiological function(s) in vascular smooth muscle and mesangial cells transfected with wild-type and carboxyl-terminal mutant receptors in vitro and transgenic mice in vivo, 2) determine the cellular and molecular regulatory mechanisms of the protein kinase-like homology domain (KHD) of NPRA and examine the effect of ANP/ATP on the phosphorylation state and signal transduction activities of NPRA, 3) examine the molecular determinants mediating the functional aspects of NPRA including the dynamics of internalization, trafficking, sequestration, and recycling by fluorescence microscopy in living cells in real time, and 4) delineate the molecular determinants mediating post-binding events and metabolic turnover including desensitization and down-regulation of NPRA in cultured VSMCs, MCs, and HEK-293 cells in vitro and transgenic mice in vivo. The proposed studies will delineate a comprehensive assessment of the mode of functioning of NPRA at the molecular level, in which detailed information about receptor structure and signaling will provide the basis for understanding receptor function and regulation. PUBLIC HEALTH RELEVANCE: Hypertension affects one billion people worldwide, and it is a potent risk factor for cardiovascular diseases. The proposed research should further our understanding of the mechanisms by which cardiac hormones regulate the blood volume and blood pressure homeostasis. Ultimately, this knowledge should yield new therapeutic targets and novel loci for the control and treatment of hypertension and cardiovascular diseases. PUBLIC HEALTH RELEVANCE: Information gained from the proposed studies will yield a more accurate assessment of the integrative role of guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) in possible mechanisms of pathogenesis whereby malregulation of receptor-mediated cardiac hormones; atrial and brain natriuretic peptides (ANP, BNP) bioactivity could result in abnormalities of fluid volume regulation and blood pressure homeostasis. Ultimately, this knowledge should yield new therapeutic molecular targets for the diagnosis, control, and treatment of hypertension and cardiovascular diseases.